Note:All cell lines are produced under aseptic conditions. Each lot is tested to be free from bacterial/fungal, mouse pathogen and mycoplasma contamination before shipping.
1. Viability is defined as the ratio of number of live cells over total number of cells after thawing
2. Immersion method is used to inoculate directly into Tryptic Soy Broth media and Fluid Thioglycollate media to check for bacteria and fungal contamination
3. PCR based assays for mouse pathogens including Sendai virus, mouse hepatitis virus, pneumonia virus, minute virus, parvovirus (MPV1,MPV2, MPV3) and Theiler’s murine encephalomyelitis virus
4. PCR based assay
5. Mouse embryonic stem cells are plated and observed for growth on a layer of feeder cells by ES cell colony morphology.
Recommended Procedure:Preparing neomycin resistant MEF as feeder cells:1. Place a vial of frozen ASF-1005 MEF cells at 37 ºC water bath immediately after removing from liquid nitrogen freezer. As soon as contents of the vial start thawing, transfer contents to a new tube containing 10X volume of pre-warmed medium
2. Spin at 1000 rpm for 5min, discard medium, resuspend cells in 8ml medium and plate cells in a T25 culture flask
3. On day 2 or 3, trypsinize cells, subculture at 1:3 ratio
4. When cells are confluent, trypsinize cells, pool and spin down, resuspend cells in growth medium, irradiate cells (gamma irradiation at 3000rad) or treat cells with mitomycin C (10 ug mitomycin C/ml for 2.5 hours)
5. Freeze down or plate treated cells at an appropriate density and they are now ready to use as feeder cells.
Suggested Drug Concentration:200ug/ml for neomycin (G418)
